The present invention relates to an oligonucleotide effective in detecting Hepatitis C virus (hereinafter, referred to as xe2x80x9cHCVxe2x80x9d) at clinical laboratory tests and diagnoses, and to a method for detecting HCV using the oligonucleotide.
HCV infection has been hitherto known as non-A non-B type infection, and is a non-oral infection caused by blood transfusion and the like. Although the HCV infection has a possibility of inducing a persistent and serious liver disease, the therapeutic method has not yet been established, so that the infection has been a big medical problem. Therefore, it is important to find out the infection in its early stages, to treat it properly, or not to use infected blood at transfusion. For the purposes, it becomes an important problem to establish an accurate and rapid detecting method at clinical laboratory tests.
As a method for determining whether the abnormality of liver function is due to HCV or not, an antibody test (enzymatic immunoadsorption method) is known. However, in such a method for detecting anti-HCV antibody, it is impossible to diagnose in its early stages before the antibody is produced. Thus, more highly effective test method is desired at actual clinical site.
HCV is a single-stranded RNA virus comprising about 9000 nucleotide, and the most sensitive test for HCV is to measure directly the virus RNA in blood. The base sequence of HCV selected from Japanese hepatitis C patients is already known (Kato et al., Proc. Natl. Acad. Sci. USA, 87, 9524-9528 (1990) and so forth). Thus, hybridization method is attempted for detection and identification of HCV using a gene probe specific to HCV RNA. In this case, the gene probe is designed so as to bind specifically to the non-translation region at the 5xe2x80x2 end or 3xe2x80x2 end of the RNA.
As described above, in antibody test, it is impossible to diagnose in its early stages of the infection, a complex operation and a long period of time are required, and also it is difficult to detect a very small amount of HCV present in a sample for a short period of time, so that it is desired to develop more rapid and highly sensitive detection method. Furthermore, in order to test more conveniently, it is required to develop an automated test apparatus.
On the other hand, for highly sensitive detection, it is preferable to detect a specific sequence of a gene to be detected and identified or an RNA derived from the gene, after their amplification.
As a method for amplifying a specific sequence of RNA of a genome such as HCV, reverse transcription-polymerase chain reaction (RT-PCR) method is known. The method comprises synthesizing CDNA of a target RNA in reverse transcription step and successively amplifying a specific DNA sequence by polymerase chain reaction wherein a cycle of thermal denaturation, primer annealing, and elongation reaction are repeatedly carried out at both ends of the specific sequence of cDNA in the presence of one pair of primers complementary to and homogeneous to the both ends and a heat-resistant DNA polymerase. However, RT-PCR method requires tedious operations, and rapid elevation and lowering of temperature are repeated, which inhibits automation of the method.
As amplification methods for a specific RNA sequences, other than RT-PCR method, there are known NASBA method, 3SR method, and the like wherein a specific RNA sequence is amplified by a synergistic action of a reverse transcriptase and an RNA polymerase. In the methods, a chain reaction is carried out wherein a double-stranded DNA containing a promoter sequence is synthesized toward a specific sequence of the target RNA by a primer containing a promoter sequence, a reverse transcriptase, and ribonuclease H, then, an RNA containing the above specific base sequence is synthesized by an RNA polymerase using the double-stranded DNA as a template, and also the RNA successively acts as a template for synthesizing the double-stranded DNA containing the promoter sequence. NASBA method or 3SR method enables nucleic acid amplification at a constant temperature, and thus, is considered to be suitable for automation. In this case, it is possible to determine the presence of HCV and also the amount thereof by qualifying or quantifying HCV RNA.
Amplification of the above specific sequence by NASBA method or the like requires an oilgonucleotide having a high specificity with the above specific sequence. Furthermore, for highly sensitive detection and identification thereof, an oligonucleotide having a high specificity with the target RNA is required. However, since the reaction is carried out at a relatively low temperature (e.g., 41xc2x0 C.) in NASBA method or the like, there is a possibility that the target RNA forms an intramolecular structure which inhibits binding of a primer and thus lowers reaction efficiency. Therefore, an operation for enhancing the binding efficiency of the primer is needed wherein the target RNA is subjected to thermal denaturation before the amplification reaction so as to break the intramolecular structure of the target RNA. Furthermore, also in the case of detecting RNA at a low temperature, an oligonucleotide capable of binding to the RNA which has formed the above intramolecular structure is required.
Thus, object of the invention is to provide an oligonucleotide useful for specific cleavage and amplification of HCV RNA, and more highly sensitive detection and identification thereof than in the conventional method, particularly, to provide an oligonucleotide useful for specific amplification of the RNA at a relatively low temperature (e.g., 41xc2x0 C.) and highly sensitive detection and identification thereof. Another object of the invention is to provide a method for amplifying the RNA using a suitable combination of such oligonucleotides and further detecting the RNA.
The first embodiment of the present invention which achieves the above-mentioned objects relates to an oligonucleotide useful for cleaving, detecting, or amplifying HCV RNA, which is capable of binding to a specific site of HCV RNA at a relatively low temperature and a constant temperature (35 to 50xc2x0 C., preferably 41xc2x0 C.) and comprises at least 10 continuous bases in any one of the sequences shown in SEQ ID NOS: 1 to 10.
The second embodiment of the invention relates to the oligonucleotide according to above first embodiment, wherein the oligonucleotide is an oligonucleotide probe for cleaving the RNA at the specific site by binding of part of the above oligonucleotide to the specific site of the RNA. The third embodiment of the invention relates to the oligonucleotide according to above first embodiment, wherein the nucleotide is an oligonucleotide primer for DNA elongation reaction. The invention of fourth embodiment of the invention relates to the oligonucleotide according to above first embodiment, wherein the nucleotide is an oligonucleotide probe modified partially or labeled with a detectable labeling substance.
The fifth embodiment of the invention relates to an amplification step which comprises:
forming an RNA-DNA duplex by forming a cDNA by the action of an RNA-dependent DNA polymerase using a specific sequence of RNA derived from HCV in a sample as a template, a first primer having a sequence homologous to the specific sequence, and a second primer having a sequence complementary to the specific sequence (wherein either primer of the first or second primers has an sequence to which a promoter sequence of an RNA polymerase is added at the 5xe2x80x2 end),
forming a single-stranded DNA by degrading the RNA-DNA duplex by ribonuclease H,
forming a double-stranded DNA having a promoter sequence capable of transcribing an RNA comprising the above RNA sequence or a sequence complementary to the above RNA sequence by a DNA-dependent DNA polymerase using the single-stranded DNA as a template, and
forming an RNA transcription product from the double-strand DNA in the presence of an RNA polymerase,
the RNA transcription product successively acting as a template for the formation of the single-stranded DNA by the above RNA-dependent DNA polymerase,
wherein the oligonucleotide of SEQ ID NO: 11 is used as the first primer and the oligonucleotide of SEQ ID NO: 6 or 7 as the second primer, the oligonucleotide of SEQ ID NO: 12 is used as the first primer and the oligonucleotide of SEQ ID NO: 7 as the second primer, or the oligonucleotide of SEQ ID NO: 13 is used as the first primer and the oligonucleotide of SEQ ID NO: 9 as the second primer.
The sixth embodiment of the invention relates to the amplification step according to above fifth embodiment, wherein the above first primer is an oligonucleotide comprising at least 10 continuous bases of the sequence of SEQ ID NO: 11, 12, or 13. The seventh embodiment of the invention relates to amplification step according to the fifth embodiment, wherein the above second primer is an oligonucleotide comprising at least 10 continuous bases of the sequence of SEQ ID NO: 6, 7, or 9. The invention of eighth embodiment of the invention relates to a method for detecting HCV RNA according to the fifth embodiment comprising measurement of fluorescent strength of a reaction solution, wherein the above RNA-amplification step is carried out in the presence of an oligonucleotide probe labeled with an intercalative fluorescent pigment, the sequence of the probe being complementary to at least part of the RNA transcription product, and the fluorescent characteristic of the probe being changed by the complementary binding to the RNA transcription product as compared with the case that the complex is not formed. The following will explain the invention in detail.